Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from Comamonas testosteroni 5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids

ABSTRACT

The invention relates to the isolation, sequencing, and recombinant expression of genes encoding either a nitrile hydratase (NHase) or amidase (Am) from  Comamonas testosteroni  5-MGAM-4D, where the NHase is useful for catalyzing the hydration of nitriles to the corresponding amides, and the amidase is useful for hydrolysis of amides to the corresponding carboxylic acids. Also provided are transformed host cells containing polynucleotides for expressing the nitrile hydratase or amidase enzymes from  Comamonas testosteroni  5-MGAM-4D.

This application claims the benefit of U.S. application Ser. No.10/977,893, filed Oct. 29, 2004, now U.S. Pat. No. 7,153,663, which is acontinuation-in-part of U.S. application Ser. No. 10/431,966, filed May8, 2003.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology andmethods for the isolation and expression of foreign genes in recombinantmicroorganisms. More specifically, the invention relates to theisolation, sequencing, and recombinant expression of nucleic acidfragments (genes) encoding either a nitrile hydratase (NHase) or amidase(Am) from Comamonas testosteroni 5-MGAM-4D, where the NHase is usefulfor catalyzing the hydration of nitriles to the corresponding amides,and the amidase is likewise useful for hydrolysis of amides to thecorresponding carboxylic acids.

BACKGROUND OF THE INVENTION

Nitrile hydratases catalyze the addition of one molecule of water to thenitrile, resulting in the formation of the corresponding amide accordingto Reaction 1:R—CN+H₂O

RCONH₂  Reaction 1

Similarly, methods for producing carboxylic acids are known and usemicroorganisms that produce an enzyme that possesses amidase (Am)activity. In general, amidases convert the amide product of Reaction 1to the corresponding carboxylic acid plus ammonia according to Reaction2:RCONH₂

RCOOH+NH₃  Reaction 2

A wide variety of bacterial genera are known to possess a diversespectrum of nitrile hydratase and amidase activities, includingRhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus,Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus(Martinkova and Kren, Biocatalysis and Biotransformation, 20:73-93(2002); Cowan et al., Extremophiles, 2:207-216 (1998)). For example,nitrile hydratase enzymes have been isolated from Pseudomonaschlororaphis B23 (Nishiyama et al., J. Bacteriol., 173:2465-2472(1991)), Rhodococcus rhodochrous J1 (Kobayashi et al., Biochem. Biophys.Acta, 1129:23-33 (1991)), Brevibacterium sp. 312 (Mayaux et al., J.Bacteriol., 172:6764-6773 (1990)), Rhodococcus sp. N-774 (Ikehata etal., Eur. J. Biochem., 181:563-570 (1989)), and Pseudomonas putida 5BNRRL-18668 (Payne et al., Biochemistry, 36:5447-5454 (1997)).

Wild-type microorganisms known to possess nitrile hydratase activityhave been used to convert nitrites to the corresponding amides. Nagasawaet al. (Appl. Microbiol. Biotechnol., 40:189-195 (1993)) have comparedthree microbial nitrile hydratase catalysts which have been used forcommercial production of acrylamide from acrylonitrile; the nitrilehydratase activities of Brevibacterium R312 and Pseudomonas chlororaphisB23 were not stable above 10° C., compared to the nitrile hydrataseactivity of Rhodococcus rhodochrous J1. Cowan et al. (supra) reportedthat many mesophilic nitrile hydratases are remarkably unstable, havingvery short enzyme activity half-lives in the growth temperature range of20-35° C. In addition to temperature instability, microbial catalystscontaining a nitrile hydratase can be susceptible to inactivation byhigh concentrations of certain substrates such as acrylonitrile. Incommercial use, the concentration of acrylonitrile was maintained at1.5-2 wt % when using Brevibacterium R312 and P. chlororaphis B23catalysts, while a concentration of up to 7 wt % was used with R.rhodochrous J1 (Nagasawa et al., supra). Similarly, Padmakumar and Oriel(Appl. Biochem. Biotechnol., 77-79:671-679 (1999)) reported thatBacillus sp. BR449 expresses a thermostable nitrile hydratase, but whenused for hydration of acrylonitrile to acrylamide, inactivation of theenzyme occurred at concentration of acrylonitrile of only 2 wt %, makingthis catalyst unsuitable for commercial applications. Webster et al.(Biotechnology Letters, 23:95-101 (2001)) compare two Rhodococcusisolates as catalysts for ammonium acrylate production (one with only anitrilase activity, and one with only a combination of nitrile hydrataseand amidase activities), and concluded that the catalyst having acombination of nitrile hydratase and amidase activities was lesspreferred due to (a) difficulty in inducing the two enzymes in therequired ratio, (b) the susceptibility of the two enzymes (nitrilehydratase and amidase) to deactivation by acrylonitrile, and (c)inhibition of the two enzymes by the respective products.

The hydration of aromatic and heteroaromatic nitrites to thecorresponding amides has been reported using the nitrile hydrataseactivity of Rhodococcus rhodochrous AJ270 (A. Meth-Cohn and M. Wang, J.Chem Soc., Perkin Trans. 1, (8):1099-1104 (1997)), where significantsubsequent conversion of the amide to the corresponding acid by amidasewas also observed. The nitrile hydratase activity of Rhodococcusrhodochrous J1 was used to convert a variety of aromatic andheteroaromatic nitrites to the corresponding amides with 100% molarconversion (J. Mauger et al., Tetrahedron, 45:1347-1354 (1989); J.Mauger et al., J. Biotechnol., 8:87-96 (1988)); an inhibitory affect ofcertain nitriles on the nitrile hydratase was overcome by maintaining alow concentration of the nitrile over the course of the reaction. U.S.20040142447 describes the use of several Rhodococcus strains for theconversion of 3-cyanopyridine to nicotinamide, where the Rhodococcusstrains were relatively stable and had a relatively low Km value for3-cyanopyridine when compared to previously-reported microbial cellcatalysts.

In addition to the use of wild-type organisms, recombinant organismscontaining heterologous genes for the expression of nitrile hydrataseare also known for the conversion of nitrites. For example, Cerebelaudet al. (WO 9504828) teach the isolation and expression in E. coli ofnitrile hydratase genes isolated from C. testosteroni. The transformedhosts effectively convert nitrites to amides, including substrates whichconsist of one nitrile and one carboxylate group. Endo et al. disclosethe production of an E. coli transformant which expresses the nitrilehydratase of Rhodococcus N-771 (U.S. Pat. No. 6,316,242 B1). Similarly,Beppu et al., (EP 5024576) disclose plasmids carrying both nitrilehydratase and amidase genes from Rhodococcus capable of transforming E.coli where the transformed host is then able to use isobutyronitrile andisobutyramide as enzymatic substrates. A stereoselective nitrilehydratase from Pseudomonas putida 5B has been overproduced in E. coli(Wuet al., Appl. Microbiol. Biotechnol., 48:704-708 (1997); U.S. Pat. No.5,811,286).

Genes encoding enzymes having amidase activity have also been cloned,sequenced, and expressed in recombinant organisms. For example, Azza etal., (FEMS Microbiol. Lett., 122:129 (1994)) disclose the cloning andover-expression in E. coli of an amidase gene from Brevibacterium sp.R312 under the control of the native promoter. Similarly, Kobayashi etal., (Eur. J. Biochem., 217:327 (1993)) teach the cloning of both anitrile hydratase and amidase gene from R. rhodococcus J1 and theirco-expression in E. coli. Wu et al. (DNA Cell Biol., 17:915-920 (1998);U.S. Pat. No. 6,251,650) report the cloning and overexpressing of a genefor amidase from Pseudomonas putida 5B in E. coli.

Applicants have previously isolated Comamonas testosteroni 5-MGAM-4D(ATCC 55744; U.S. Pat. No. 5,858,736 and U.S. Pat. No. 5,922,589).Comamonas testosteroni 5-MGAM-4D has been shown to containthermally-stable, regiospecific nitrile hydratase (EC 4.2.1.84) andamidase (EC 3.5.1.4) activities useful in the conversion of a variety ofnitriles to their corresponding amides and carboxylic acids. Methodsillustrating the utility of the Comamonas testosteroni 5-MGAM-4D nitrilehydratase and amidase activities have been described previously by theApplicants. These uses include regio-selective preparation of lactamsfrom aliphatic α,ω-dinitriles (U.S. Pat. No. 5,858,736), bioconversionof 3-hydroxynitriles to 3-hydroxyacids (U.S. 2002/0039770 A1), andbioconversion of methacrylonitrile and acrylonitrile to theircorresponding carboxylic acids (U.S. Ser. No. 10/067,652), herebyincorporated by reference. However, the isolation and recombinantexpression of the nucleic acid fragments encoding the nitrile hydrataseand amidase from Comamonas testosteroni 5-MGAM-4D has been elusive.

The problem to be solved is to provide the genes and encoding for thethermally-stable, regio-selective nitrile hydratase and amidase enzymesfrom Comamonas testosteroni 5-MGAM-4D and to provide transformantsexpressing these catalysts.

Additionally, the development of industrial processes which employmicrobial catalysts having nitrile hydratase/amidase activities toefficiently manufacture amides or carboxylic acids has proved difficult.Many methods using enzyme catalysts to prepare these products from thecorresponding nitrites do not produce and accumulate the product at asufficiently high concentration to meet commercial needs, or are subjectto enzyme inactivation (requiring a low concentration of nitrile overthe course of the reaction) or product inhibition during the course ofthe reaction.

The additional problem to be solved continues to be the lack of facilemicrobial catalysts to convert nitrites to the corresponding amides oracids in a process characterized by high yield, high concentration, andhigh selectivity, and with the added advantages of low temperature andenergy requirements and low waste production when compared to knownchemical methods of nitrile hydrolysis. Comamonas testosteroni 5-MGAM-4Dexpresses a thermally-stable, regio-selective nitrile hydratase as wellas a thermally-stable amidase. An enzyme catalyst having only thenitrile hydratase activity of Comamonas testosteroni 5-MGAM-4D would behighly useful in applications where only the amide product from nitrilehydration is desired.

SUMMARY OF THE INVENTION

The Applicants have isolated and sequenced the genes necessary toexpress thermally-stable, regio-selective nitrile hydratase and amidasefrom Comamonas testosteroni 5-MGAM4D. The corresponding amino acidsequences for each enzyme are also disclosed. The invention alsoencompasses 1) an isolated polynucleotide encoding a polypeptide havingat least 98% identity to a polypeptide alpha-subunit of the nitrilehydratase enzyme from Comamonas testosteroni 5-MGAM-4D as represented bySEQ ID No:4; 2) an isolated polynucleotide encoding a polypeptide havingat least 95% identity to a polypeptide beta-subunit of the nitrilehydratase enzyme from Comamonas testosteroni 5-MGAM-4D as represented bySEQ ID No:6; and 3) an isolated polynucleotide encoding a polypeptidehaving amidase activity and having at least 95% identity to thepolypeptide from Comamonas testosteroni 5-MGAM4D as represented by SEQID No:17.

The invention further provides a region of the Comamonas testosteroni5-MGAM-4D genome encompassed within a 0.9 kb fragment (SEQ ID NO:9)which encodes a polypeptide (designated “PK7”; and represented by SEQ IDNO:14) that is necessary for optimum activity of the nitrile hydrataseenzyme.

Transformants are provided which express either the nitrile hydratase oramidase enzymes separately or which co-express both enzymes. Alsoprovided are methods to produce the nitrile hydratase and amidasecatalysts in a recombinant host. The present invention further providesrecombinant hosts, transformed with the polynucleotides encoding theamidase and/or the nitrile hydratase in combination with the PK7accessory protein.

A particular embodiment of the invention is Escherichia coli transformedwith the nucleic acid sequence represented by SEQ ID NO:11.

The Applicants also provide methods for converting a variety ofaliphatic nitrites, aromatic nitrites, heterocyclic aromatic nitrites,unsaturated nitrites, aliphatic dinitriles, and 2-, 3-, or4-hydroxynitriles to the corresponding amides using a transformed hostcell expressing the nitrile hydratase from Comamonas testosteroni5-MGAM-4D. Comamonas testosteroni 5-MGAM-4D expresses athermally-stable, regioselective nitrile hydratase as well as athermally-stable amidase. The present application describes thepreparation of microbial transformants that have only the nitrilehydratase activity of Comamonas testosteroni 5-MGAM-4D for use inapplications where the production of only the amide product from nitrilehydration would be desirable.

BRIEF DESCRIPTION OF THE DRAWINGS, SEQUENCE DESCRIPTIONS, AND BIOLOGICALDEPOSITS

The invention can be more fully understood from the Figure, the SequenceListing, the Biological Deposits, and the detailed description thattogether form this application.

FIG. 1 shows the nucleic acid fragments inserted in several plasmidscreated for recombinant expression of genes cloned from Comamonastestosteroni 5-MGAM-4D (ATCC 55744).

The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirementsfor Patent Applications Containing Nucleotide Sequences and/or AminoAcid Sequence Disclosures—the Sequence Rules”) and are consistent withWorld Intellectual Property Organization (WIPO) Standard ST.25 (1998)and the sequence listing requirements of the EPO and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the nucleic acid sequence encoding the α-subunit of anitrile hydratase from Pseudomonas putida 5B (NRRL-18668) used to probegenomic DNA fragments from Comamonas testosteroni 5-MGAM-4D (ATCC55744).

SEQ ID NO:2 is the nucleic acid sequence encoding the β-subunit of anitrile hydratase from Pseudomonas putida 5B (NRRL-18668) used to probegenomic DNA fragments from Comamonas testosteroni 5-MGAM-4D (ATCC55744).

SEQ ID NO:3 is the nucleic acid sequence encoding the α-subunit of anitrile hydratase from Comamonas testosteroni 5-MGAM-4D.

SEQ ID NO:4 is the deduced amino acid sequence for the α-subunit of anitrile hydratase from Comamonas testosteroni 5-MGAM-4D.

SEQ ID NO:5 is the nucleic acid sequence encoding the β-subunit of anitrile hydratase from Comamonas testosteroni 5-MGAM-4D.

SEQ ID NO:6 is the deduced amino acid sequence for the β-subunit of anitrile hydratase from Comamonas testosteroni 5-MGAM-4D.

SEQ ID NO:7 is the nucleic acid sequence encoding the α- and β-subunitsof the nitrile hydratase from Comamonas testosteroni 5-MGAM-4D used inthe creation of pSW131.

SEQ ID NO:8 is the first of two primers (“Primer 1”) useful foramplifying a nucleic acid fragment (SEQ ID NO:7) for creation of plasmidpSW131 and for amplifying a nucleic acid fragment (SEQ ID NO:11) forcreation of pSW137.

SEQ ID NO:9 is the second of two primers (“Primer 2”) useful foramplifying a nucleic acid fragment (SEQ ID NO:7) for creation of plasmidpSW131.

SEQ ID NO:10 is the nucleic acid sequence of a 0.9 kb nucleic acidfragment from Comamonas testosteroni 5-MGAM-4D containing a small openreading frame (ORF) which encodes an accessory protein (denoted as“P7K”) useful in the expression of active nitrile hydratase.

SEQ ID NO:11 is the nucleic acid sequence encoding the α- and β-subunitsof the nitrile hydratase plus 0.9 kb of downstream DNA (SEQ ID NO. 10)encoding the accessory protein P7K from Comamonas testosteroni 5-MGAM-4Dused in the creation of pSW132 and pSW137.

SEQ ID NO:12 is the second of two primers (“Primer 3”) useful foramplifying a nucleic acid fragment (SEQ ID NO:11) for creation of pSW137and for amplifying a nucleic acid fragment (SEQ ID NO:23) for creationof pSW136.

SEQ ID NO:13 is the nucleic acid sequence encoding the accessory proteinP7K, and found within the 0.9 kb downstream DNA sequence (SEQ ID NO:10)from Comamonas testosteroni 5-MGAM-4D.

SEQ ID NO:14 is the deduced amino acid sequence for the accessoryprotein P7K useful in the recombinant expression of Comamonastestosteroni 5-MGAM-4D nitrile hydratase.

SEQ ID NO:15 is the nucleic acid sequence of a nucleic acid fragmentcomprising the first 0.6 kb of the pKP57 insert useful as a probe toidentify an amidase from Comamonas testosteroni 5-MGAM-4D.

SEQ ID NO:16 is the nucleic acid sequence encoding an amidase fromComamonas testosteroni 5-MGAM-4D.

SEQ ID NO:17 is the deduced amino acid sequence of an amidase fromComamonas testosteroni 5-MGAM4D.

SEQ ID NO:18 is the nucleic acid sequence of 7.4 kb nucleic acidfragment comprising the complete coding sequences for an amidase and anitrile hydratase and the P7K accessory protein from Comamonastestosteroni 5-MGAM-4D.

SEQ ID NO:19 is the first of two primers (“Primer 4”) useful foramplifying a nucleic acid fragment encoding an amidase from Comamonastestosteroni 5-MGAM-4D for creation of plasmid pKP60.

SEQ ID NO:20 is the second of two primers (“Primer 5”) useful foramplifying a nucleic acid fragment encoding an amidase from Comamonastestosteroni 5-MGAM-4D for creation of plasmid pKP60.

SEQ ID NO:21 is the first of two primers (“Primer 6”) useful foramplifying a nucleic acid fragment encoding an amidase from Comamonastestosteroni 5-MGAM-4D for creation of plasmid pSW133 and for amplifyinga nucleic acid fragment (SEQ ID NO:23) for creation of pSW136.

SEQ ID NO:22 is the second of two primers (“Primer 7”) useful foramplifying a nucleic acid fragment encoding an amidase from Comamonastestosteroni 5-MGAM-4D for creation of plasmid pSW133.

SEQ ID NO:23 is the nucleic acid fragment encoding an amidase, a nitrilehydratase (α- and β-subunits), and the accessory protein P7K fromComamonas testosteroni 5-MGAM-4D used in the creation of plasmid pSW136.

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure:

Depositor Identification Int'l. Depository Reference Designation Date ofDeposit Comamonas testosteroni ATCC 55744 Mar. 8, 1996 5-MGAM-4DPseudomonas putida 5B NRRL 18668 Jul. 6, 1990 Escherichia coli SW132ATCC PTA-5073 Mar. 21, 2003 Escherichia coli SW137 ATCC PTA-5074 Mar.21, 2003

As used herein, “ATCC” refers to the American Type Culture CollectionInternational Depository Authority located at ATCC, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. The “International DepositoryDesignation” is the accession number to the culture on deposit withATCC.

As used herein, “NRRL” refers to the Northern Regional ResearchLaboratory, Agricultural Research Service Culture CollectionInternational Depository Authority located at 11815 N. UniversityStreet, Peoria, Ill. 61604 U.S.A. The “NRRL No.” is the accession numberto cultures on deposit at the NRRL.

The listed deposits will be maintained in the indicated internationaldepository for at least thirty (30) years and will be made available tothe public upon the grant of a patent disclosing it. The availability ofa deposit does not constitute a license to practice the subjectinvention in derogation of patent rights granted by government action.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated polynucleotides and the nucleicacid sequences that encode three polypeptides (α- and β-subunits of anitrile hydratase and an amidase) from Comamonas testosteroni 5-MGAM-4D(ATCC 55744) that act as catalysts. When coexpressed, the α- andβ-subunits of the nitrile hydratase selectively hydrate nitrites intothe corresponding amides, and the amidase hydrolyzes amides into thecorresponding carboxylic acids. The invention also provides transformedmicrobial host cells expressing the polypeptides. The invention furtherprovides a method for producing the polypeptide catalysts using thetransformed microbes and a method for using the catalysts for convertingnitrites to the corresponding amides and/or carboxylic acids, or forconverting amides to the corresponding carboxylic acids.

Definitions:

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

The terms “catalyst”, “enzyme catalyst” or “microbial cell catalyst”refer to polypeptides (or proteins) having a nitrile hydratase activity,an amidase activity, or having a combination of nitrile hydratase andamidase activities. The catalyst may be in the form of an intactmicrobial cell, permeabilized microbial cell(s), one or more cellcomponents of a microbial cell extract, partially purified enzyme(s), orpurified enzyme(s).

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

The term “thermally-stable” characterizes an enzyme that retainsactivity despite exposure to a given temperature.

The terms “Comamonas testosteroni” and “C. testosteroni” are usedinterchangeably and refer to Comamonas testosteroni 5-MGAM-4D (ATCC55744).

The terms “Psuedomonas putida 5B” and “P. putida 5B” are usedinterchangeably and refer to Psuedomonas putida NRRL-18668.

The terms “Escherichia coli SW132” and “E. coli SW132” are usedinterchangeably and refer to an E. coli strain transformed with plasmidpSW132 and having ATCC accession number PTA-5073.

The term “pSW132” refers to a plasmid containing a DNA fragment encodingthe C. testosteroni 5-MGAM-4D nitrile hydratase α- and β-subunits plus0.9 kb of downstream DNA under the control of the T7 promoter. The 0.9kb of downstream DNA encodes an accessory protein (“P7K”) useful inrecombinant expression of nitrile hydratase. E. coli strain SW132harbors plasmid pSW132 and has ATCC accession number PTA-5073.

The terms “Escherichia coli SW137” and “E. coli SW137” are usedinterchangeably and refer to an E. coli strain transformed with plasmidpSW137 and having ATCC accession number PTA-5074.

The term “pSW137” refers to a plasmid containing a DNA fragment encodingthe C. testosteroni 5-MGAM-4D nitrile hydratase α- and β-subunits plus0.9 kb of downstream DNA under the control of the trc promoter. The 0.9kb of downstream DNA encodes an accessory protein (“P7K”) useful inrecombinant expression of nitrile hydratase. E. coli strain SW137harbors plasmid pSW137 and has ATCC accession number PTA-5074.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

As used herein, an “isolated nucleic acid fragment” or “isolatedpolynucleotide” is a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural, oraltered nucleotide bases. An isolated nucleic acid fragment in the formof a polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, or synthetic DNA.

The term “accessory nucleic acid” refers to the 0.9 kb sequence (SEQ IDNO:10), located downstream of the nitrile hydratase α (alpha) and β(beta) subunit genes, which contains an open reading frame (SEQ IDNO:13) encoding a polypeptide (SEQ ID NO:14) useful in increasedrecombinant expression of the nitrile hydratase.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Preferred methods to determine identity are designedto give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences may be performed using the CLUSTAL method ofalignment (Higgins and Sharp CABIOS. 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameterstypically used for pairwise alignments using the CLUSTAL method areKTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the instant microbialpolypeptides as set forth in SEQ ID NOs:4, 6, 14, and 17. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. In the present invention, the host cell's genome includeschromosomal and extrachromosomal (e.g. plasmid) genes. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” or “recombinant” or “transformed” organisms.

The term “carbon substrate” refers to a carbon source capable of beingmetabolized by host organisms of the present invention, and particularlyrefers to carbon sources selected from, but not limited to, the groupconsisting of aliphatic carboxylic acids or dicarboxylic acids,monosaccharides, oligosaccharides, polysaccharides, and one-carbonsubstrates or mixtures thereof.

Nitriles particularly pertinent to the invention are nitriles ofR—C≡N  Formula 1orN≡C—R—C≡N  Formula 2wherein N is Nitrogen, C is Carbon, and R is selected from the groupconsisting of: a) C₁-C₉ alkyl, that is linear, branched or cyclic,optionally substituted with a hydroxyl group, an amino group, acarboxylic acid group, a carboxamide group, a halogen atom, or an oxogroup; b) C₁-C₉ alkenyl, linear, branched or cyclic, optionallysubstituted with a hydroxyl group, an amino group, a carboxylic acidgroup, a carboxamide group, a halogen atom, or an oxo group; and c) C₄C₆-C₉ aryl, optionally substituted with a hydroxyl group, an aminogroup, a carboxylic acid group, a carboxamide group, or a halogen atom.Particularly useful nitriles in the invention include, but are notlimited to, acrylonitrile, methacrylonitrile, 3-hydroxypropionitrile,3-hydroxybutyronitrile, 3-hydroxyvaleronitrile, butyronitrile,adiponitrile, benzonitrile, and glycolonitrile.

Additional nitriles particularly pertinent to the invention are nitrilesof Formula 3:R²—C≡N  Formula 3wherein N is Nitrogen, C is Carbon, and R² is selected from the groupconsisting of the general formulas 4, 5, 6, 7 and 8:

where in Formula 4, 5 and 6 X is N, in Formula 7 X is NH, O or S, and inFormula 8 X is NH and Y is N, O or S, and where R³ and R⁴ are,independently, selected from the group comprised of: a) a hydrogen atom,b) a halogen atom, c) a C₁-C₉ alkyl group that is linear, branched orcyclic and optionally substituted with a hydroxyl group, an amino group,a carboxylic acid group, a carboxamide group, a halogen atom, or an oxogroup, d) a C₁-C₉ alkenyl group that is linear, branched or cyclic andoptionally substituted with a hydroxyl group, an amino group, acarboxylic acid group, a carboxamide group, a halogen atom, or an oxogroup, and e) a C₆-C₉ aryl, optionally substituted with a hydroxylgroup, an amino group, a carboxylic acid group, a carboxamide group, ora halogen atom. Halogen atoms may be independently F, Cl, Br, or I.Particularly useful heterocyclic nitriles in the invention include, butare not limited to, 3-cyanopyridine, 4-cyanopyridine,pyrazinecarbonitrile, 2-furancarbonitrile, 2-thiophenecarbonitrile, and4-thiazolecarbonitrile.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “altered biological activity” will refer to an activity,associated with a polypeptide (protein) encoded by a microbialnucleotide sequence which can be measured by an assay method, where thatactivity is either greater than or less than the activity associatedwith the native microbial sequence. “Enhanced biological activity”refers to an altered activity that is greater than that associated withthe native sequence. “Diminished biological activity” is an alteredactivity that is less than that associated with the native sequence.

The terms “suitable aqueous reaction mixture” or “suitable reactionmixture” refer to the materials and water in which the nitrile and/oramide substrate and enzyme catalyst come into contact. Components ofsuitable aqueous reaction mixtures are referred to herein and thoseskilled in the art appreciate the range of component variations suitablefor this process.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA), and the FASTA program incorporating the Smith-Waterman algorithm(W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994),Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum,New York, N.Y.). The term “MEME” refers to a software program used toidentify conserved diagnostic motifs based on a hidden Markov model(Timothy L. Bailey and Charles Elkan, Fitting a mixture model byexpectation maximization to discover motifs in biopolymers, Proceedingsof the Second International Conference on Intelligent Systems forMolecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif. (1994)).“MAST” (Timothy L. Bailey and Michael Gribskov, “Combining evidenceusing p-values: application to sequence homology searches”Bioinformatics, Vol. 14, pp. 48-54 (1998)) is a program that takes theoutput from the MEME program and searches the identified motifs againstthe protein databases such as EMBL and SwissProt. Within the context ofthis application it will be understood that where sequence analysissoftware is used for analysis, that the results of the analysis will bebased on the “default values” of the program referenced, unlessotherwise specified. As used herein “default values” will mean any setof values or parameters which originally load with the software whenfirst initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984) (hereinafter“Silhavy”); and by Ausubel, F. M. et al., Current Protocols in MolecularBiology, published by Greene Publishing Assoc. and Wiley-Interscience(1987) (hereinafter “Ausubel”).

Sequence Identification

Comparison of the present amino acid sequences for the nitrile hydrataseα and β-subunits and the amidase from Comamonas testosteroni 5-MGAM-4Dto public databases revealed that the most similar-known sequences wereall from Pseudomonas putida 5B (NRRL-18668; see: U.S. Pat. No.5,811,286, and Wu et al., Appl. Microbiol. Biotechnol. 48:704-708(1997)). The nitrile hydratase α-subunit was about 97.1% identical tothe corresponding nitrile hydratase α-subunit from P. putida 5B (Table1). The nitrile hydratase β-subunit was about 82.0% identical to thecorresponding nitrile hydratase β-subunit from P. putida 5B. Lastly, theamidase was about 92.3% identical to the corresponding amidase from P.putida 5B.

TABLE 1 Sequence Analysis Results Gene % % Name Similarity IdentifiedIdentity^(a) Similarity^(b) E-value^(c) Citation AmidaseGi|6225048|sp|O69768| 92.3 92.5 0 Wu et al., DNA Cell AMID_PSEPU Biol.17 (10), 915-920 (1998) nitrile Gi|2499193|sp|P97051 97.1 97.1 10e−108Payne et al., hydratase |NHAA_PSEPU Biochemistry 36 (18), α-subunit5447-5454 (1997) nitrile Gi|2499195|sp|P97052| 82.0 82.5 6e−92 Payne etal., hydratase NHAB_PSEPU Biochemistry 36 (18), β-subunit 5447-5454(1997) ^(a)Identity is defined as percentage of amino acids that areidentical between the two proteins. ^(b)Similarity is defined aspercentage of amino acids that are identical or conserved between thetwo proteins. ^(c)Expect value. The Expect value estimates thestatistical significance of the match, specifying the number of matches,with a given score, that are expected in a search of a database of thissize absolutely by chance.

Despite the sequence similarities between the nitrile hydratase andamidase enzymes of Comamonas testosteroni 5-MGAM4D to those ofPseudomonas putida 5B (NRRL-18668), the accompanying Examplesdemonstrate that both the thermal stability, and stability underreaction conditions, of the Comamonas testosteroni 5-MGAM4D nitrilehydratase enzyme (expressed in E. coli transformant SW132) are bothdifferent and markedly superior to the Pseudomonas putida 5B nitrilehydratase (expressed in E. coli transformant SW30, Wu et al., supra).

Identification of Homologs

The instant sequences may be employed as hybridization reagents for theidentification of homologs. The basic components of a nucleic acidhybridization test include a probe, a sample suspected of containing thegene or gene fragment of interest, and a specific hybridization method.Probes of the present invention are typically single stranded nucleicacid sequences which are complementary to the nucleic acid sequences tobe detected. Probes are “hybridizable” to the nucleic acid sequence tobe detected. The probe length can vary from 5 bases to tens of thousandsof bases, and will depend upon the specific test to be done. Typically aprobe length of about 15 bases to about 30 bases is suitable. Only partof the probe molecule need be complementary to the nucleic acid sequenceto be detected. In addition, the complementarity between the probe andthe target sequence need not be perfect. Hybridization does occurbetween imperfectly complementary molecules with the result that acertain fraction of the bases in the hybridized region are not pairedwith the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration theshorter the hybridization incubation time needed. Optionally achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.,19:5143-5151, (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide, and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers,such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9),about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between0.5-20 mM EDTA, FICOLL (about 300-500 kilodaltons), polyvinylpyrrolidone(about 250-500 kdal), and serum albumin. Also included in the typicalhybridization solution will be unlabeled carrier nucleic acids fromabout 0.1 to 5 mg/mL, fragmented nucleic DNA, e.g., calf thymus orsalmon sperm DNA, or yeast RNA, and optionally from about 0.5 to 2%wt./vol. glycine. Other additives may also be included, such as volumeexclusion agents which include a variety of polar water-soluble orswellable agents, such as polyethylene glycol, anionic polymers such aspolyacrylate or polymethylacrylate, and anionic saccharidic polymers,such as dextran sulfate.

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Microbial Recombinant Expression

The genes and gene products of the instant sequences may be produced inheterologous host cells, particularly in the cells of microbial hosts.Expression in recombinant microbial hosts may be useful for theexpression of various pathway intermediates; for the modulation ofpathways already existing in the host, or for the synthesis of newproducts heretofore not possible using the host.

Preferred heterologous host cells for expression of the instant genesand nucleic acid fragments are microbial hosts that can be found broadlywithin the fungal or bacterial families and which grow over a wide rangeof temperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi will besuitable hosts for expression of the present nucleic acid fragments.Because of transcription, translation and the protein biosyntheticapparatus is the same irrespective of the cellular feedstock, functionalgenes are expressed irrespective of carbon feedstock used to generatecellular biomass. Large-scale microbial growth and functional geneexpression may utilize a wide range of simple or complex carbohydrates,organic acids and alcohols, saturated hydrocarbons such as methane orcarbon dioxide in the case of photosynthetic or chemoautotrophic hosts.However, the functional genes may be regulated, repressed or depressedby specific growth conditions, which may include the form and amount ofnitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrientincluding small inorganic ions. In addition, the regulation offunctional genes may be achieved by the presence or absence of specificregulatory molecules that are added to the culture and are not typicallyconsidered nutrient or energy sources.

Examples of host strains include but are not limited to bacterial,fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter,Zymomonas, Agrobacterium, Erythrobacter Chlorobium, Chromatium,Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces,Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia,Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas,Methylobacter, Methylococcus, Methylosinus, Methylomicrobium,Methylocystis, Methylobacterium, Alcaligenes, Synechocystis,Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella,Myxococcus, and Staphylococcus. In another embodiment, suitable hoststrains are selected from the group consisting of Aspergillus,Saccharomyces, Pichia, Candida, Hansuela, Bacillus, Rhodococcus,Streptomyces, Brevibacterium, Corynebacteria, Escherichia, Pseudomonas,Methylomonas, Synechocystis, and Klebsiella. In a further embodiment,suitable host strains are selected from the group consisting ofBacillus, Rhodococcus, Escherichia, Pseudomonas, Klebsiella, andMethylomonas.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for expression of the presentnitrile hydratase, amidase, and PK7. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the enzymes

Accordingly it is expected, for example, that introduction of chimericgenes encoding the instant bacterial enzyme under the control of theappropriate promoter, will demonstrate increased nitrile to amide and/orcarboxylic acid conversion. It is contemplated that it will be useful toexpress the instant genes both in natural host cells as well as in aheterologous host. Introduction of the present genes into native hostswill result in altered levels of existing nitrile hydratase and amidaseactivity. Additionally, the instant genes may also be introduced intonon-native host bacteria where an existing nitrile-amide-carboxylic acidpathway may be manipulated.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the instant ORF in the desired host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdriving these genes is suitable for the present invention including butnot limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1,TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces);AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IP_(L),IP_(R), T7, tac, and trc (useful for expression in Escherichia coli) aswell as the amy, apr, npr promoters and various phage promoters usefulfor expression in Bacillus. Additionally, the deoxy-xylulose phosphatesynthase or methanol dehydrogenase operon promoter (Springer et al.,FEMS Microbiol Lett 160:119-124 (1998)), the promoter forpolyhydroxyalkanoic acid synthesis (Foellner et al., Appl. Microbiol.Biotechnol. 40:284-291 (1993)), promoters identified from nativeplasmids in methylotrophs (EP 296484), promoters identified frommethanotrophs (PCT/US03/33698), and promoters associated with antibioticresistance [e.g., kanamycin (Springer et al., supra; Ueda et al., Appl.Environ. Microbiol 57:924-926 (1991)) or tetracycline (U.S. Pat. No.4,824,786)] are suitable for expression of the present coding sequences,especially in C1 metabolizers.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

Methods of manipulating genetic pathways are common and well known inthe art. Selected genes in a particularly pathway may be up-regulated ordown-regulated by variety of methods. Additionally, competing pathwaysmay be eliminated or sublimated by gene disruption and similartechniques.

Once a key genetic pathway has been identified and sequenced, specificgenes may be up-regulated to increase the output of the pathway. Forexample, additional copies of the targeted genes may be introduced intothe host cell on multicopy plasmids such as pBR322. Alternatively thetarget genes may be modified so as to be under the control of non-nativepromoters. Where it is desired that a pathway operate at a particularpoint in a cell cycle or during a fermentation run, regulated orinducible promoters may used to replace the native promoter of thetarget gene. Similarly, in some cases the native or endogenous promotermay be modified to increase gene expression. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868).

Alternatively, it may be necessary to reduce or eliminate the expressionof certain genes in a pathway or in competing pathways that may serve ascompeting sinks for energy or carbon. Methods of down-regulating genesfor this purpose have been explored. Where sequence of the gene to bedisrupted is known, one of the most effective methods of gene downregulation is targeted gene disruption where foreign DNA is insertedinto a structural gene so as to disrupt transcription. This can beeffected by the creation of genetic cassettes comprising the DNA to beinserted (often a genetic marker) flanked by sequence having a highdegree of homology to a portion of the gene to be disrupted.Introduction of the cassette into the host cell results in insertion ofthe foreign DNA into the structural gene via the native DNA replicationmechanisms of the cell (Hamilton et al., J. Bacteriol. 171:4617-4622(1989); Balbas et al., Gene 136:211-213 (1993); Gueldener et al.,Nucleic Acids Res. 24:2519-2524 (1996); and Smith et al., Methods Mol.Cell. Biol. 5:270-277(1996)).

Antisense technology is another method of down regulating genes wherethe sequence of the target gene is known. To accomplish this, a nucleicacid segment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA which encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence based. For example, cells may be exposed to a UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect non-replicating DNA such asHNO₂ and NH₂OH, as well as agents that affect replicating DNA such asacridine dyes, notable for causing frameshift mutations. Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. (See for example, Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass. (hereinafter“Brock”), or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227(1992) (hereinafter “Deshpande”)).

Another non-specific method of gene disruption is the use oftransposable elements or transposons. Transposons are genetic elementsthat insert randomly in DNA but can be latter retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon, is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutagenesis and for gene isolation, since thedisrupted gene may be identified on the basis of the sequence of thetransposable element. Kits for in vitro transposition are commerciallyavailable (see for example The Primer Island Transposition Kit,available from Perkin Elmer Applied Biosystems, Branchburg, N.J., basedupon the yeast Ty1 element; The Genome Priming System, available fromNew England Biolabs, Beverly, Mass.; based upon the bacterial transposonTn7; and the EZ::TN Transposon Insertion Systems, available fromEpicentre Technologies, Madison, Wis., based upon the Tn5 bacterialtransposable element).

Industrial Production of Biocatalyst

Commercial production of biocatalyst for preparing amides usingtransformants harboring the nitrile hydratase catalyst disclosed herein(encoded by genes for the α- and β-subunits, and optionally, accessoryprotein P7K) and for preparing carboxylic acids from amides using theamidase catalyst disclosed herein may be conducted using a variety ofculture methodologies. Large-scale production of a specific gene productmay be produced by both batch and continuous culture methodologies.

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media. Measurement of the actual substrateconcentration in Fed-Batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases such as CO₂.Batch and Fed-Batch culturing methods are common and well known in theart and examples may be found in Brock (supra) and Deshpande (supra).

Commercial production of biocatalysts may also be accomplished with acontinuous culture. Continuous cultures are an open system where adefined culture media is added continuously to a bioreactor and an equalamount of conditioned media is removed simultaneously for processing.Continuous cultures generally maintain the cells at a constanthigh-liquid-phase density where cells are primarily in log phase growth.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited toaliphatic carboxylic acid and dicarboxylic acids (such as lactic acid orsuccinic acid), glycerol, monosaccharides (such as glucose andfructose), disaccharides (such as lactose or sucrose), oligosaccharides(such as soluble starch), polysaccharides (such as starch or celluloseor mixtures thereof), and unpurified mixtures from renewable feedstocks(such as cheese whey permeate, cornsteep liquor, sugar beet molasses,and barley malt). Additionally the carbon substrate may also beone-carbon substrates such as carbon dioxide, methane or methanol forwhich metabolic conversion into key biochemical intermediates has beendemonstrated. In addition to one and two carbon substrates,methylotrophic organisms are also known to utilize a number of othercarbon-containing compounds such as methylamine, glucosamine, and avariety of amino acids for metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.,[Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153:485-489 (1990)). Therefore, it is contemplated that thesource of carbon utilized in the present invention may encompass a widevariety of carbon-containing substrates and will only be limited by themicroorganism employed.

Biocatalytic Conversion of Nitriles to Amides or Carboxylic Acids

An aqueous reaction mixture containing the aliphatic, aromatic, orheterocyclic aromatic nitrile is prepared by mixing the nitrile with anaqueous suspension of the appropriate enzyme catalyst. Intact microbialcells can be used as catalyst without any pretreatment, such aspermeabilization or heating. Alternatively, the cells can be immobilizedin a polymer matrix (e.g., alginate, carrageenan, polyvinyl alcohol, orpolyacrylamide gel (PAG)) or on a soluble or insoluble support (e.g.,celite, silica) to facilitate recovery and reuse of the catalyst.Methods to immobilize cells in a polymer matrix or on a soluble orinsoluble support have been widely reported and are well known to thoseskilled in the art. The enzyme can also be isolated from the microbialcells and used directly as catalyst, or the enzyme can be immobilized ina polymer matrix or on a soluble or insoluble support. These methodshave also been widely reported and are well known to those skilled inthe art (Methods in Biotechnology, Vol. 1: Immobilization of Enzymes andCells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, N.J., USA;1997).

Intact microbial cells, either immobilized or unimmobilized, containinggenes that encode a polypeptide having nitrile hydratase activity oramidase activity, or containing genes that encode a combination ofpolypeptides separately having nitrile hydratase and amidase activities,can be used as catalyst without any pretreatment, such aspermeabilization, freeze thawing or heating. Alternatively, themicrobial cells may be permeabilized by methods familiar to thoseskilled in the art (e.g., treatment with toluene, detergents, orfreeze-thawing) to improve the rate of diffusion of materials into andout of the cells. Methods for permeabilization of microbial cells arewell-known to those skilled in the art (Felix, H., Anal. Biochem.,120:211-234 (1982)).

Some of the aliphatic or aromatic nitriles used as starting material inthe present invention are only moderately water soluble. Theirsolubility also depends on the temperature of the solution and the saltconcentration in the aqueous phase; the optional inclusion of a buffer,or the production of the ammonium salt of a carboxylic acid byhydrolysis of the corresponding amide are two possible sources of saltin a reaction mixture. In the present case, producing a hydrated orhydrolyzed reaction product at a concentration greater than thesolubility limit of the starting aliphatic or aromatic nitrile isaccomplished using a reaction mixture that is initially composed of twophases: an aqueous phase (containing the enzyme catalyst and dissolvedaliphatic or aromatic nitrile) and an organic phase (the undissolvedaliphatic or aromatic nitrile, optionally dissolved in an organicsolvent not miscible with the aqueous phase). As the reactionprogresses, the aliphatic or aromatic nitrile dissolves into the aqueousphase, eventually yielding a product mixture which may be a singlephase, depending on the solubility of the products in water, and on thepresence or absence of an optional organic solvent not miscible withwater.

The aqueous phase of a two-phase reaction mixture can contain, at aminimum, only as much water as is sufficient to result in a) completeconversion of the aliphatic or aromatic nitrile to the correspondingamide or carboxylic acid (dependent on whether only active nitrilehydratase or a combination of active nitrile hydratase and amidaseenzyme are present), and b) maintenance of the hydrolytic activity ofthe enzyme catalyst. The reaction may also be run by adding thealiphatic or aromatic nitrile to the reaction mixture at a rateapproximately equal to the enzymatic hydration or hydrolysis reactionrate, thereby maintaining a single-phase aqueous reaction mixture,thereby avoiding the potential problem of substrate inhibition of theenzyme at high starting material concentrations.

The final concentration of aliphatic or aromatic amide or carboxylicacid in solution in the product mixture at complete conversion of thecorresponding aliphatic or aromatic nitrile may range from 0.001 M tothe solubility limit of the aliphatic or aromatic nitrile in the productmixture. Product may precipitate from the reaction mixture during thecourse of the reaction, allowing for the production of amide orcarboxylic acid in excess of the solubility of said product in thereaction mixture. Typically, the concentration of the aliphatic oraromatic amide or carboxylic acid product in solution in the productmixture ranges from 0.001 M to 7.0 M. The aliphatic or aromatic amide orcarboxylic acid may also be isolated from the product mixture (afterremoval of the catalyst) by optionally adjusting the pH of the reactionmixture to between 2.0 and 2.5 with concentrated HCl when the product ofthe reaction is a carboxylic acid, saturating the resulting solutionwith sodium chloride, and extracting the aliphatic or aromatic amide orcarboxylic acid with a suitable organic solvent, such as ethyl acetate,ethyl ether, methyl isobutyl ketone or dichloromethane. The organicextracts are then combined, stirred with a suitable drying agent (e.g.,magnesium sulfate), filtered, and the solvent removed (e.g., by rotaryevaporation) to produce the desired product in high yield and in highpurity (typically 98-99% pure). If desired, the product can be furtherpurified by recrystallization or distillation.

The concentration of enzyme catalyst in the reaction mixture depends onthe specific catalytic activity of the enzyme catalyst and is chosen toobtain the desired rate of reaction. The wet cell weight of themicrobial cells used as catalyst in hydrolysis reactions typicallyranges from 0.001 grams to 0.300 grams of wet cells per mL of totalreaction volume, preferably from 0.002 grams to 0.050 grams of wet cellsper mL; the cells may be optionally immobilized as described above. Thespecific activity of the microbial cells (IU/gram dry cell weight) isdetermined by measuring the rate of conversion of a 0.10-0.50 M solutionof a nitrile substrate to the desired amide or carboxylic acid productat 25° C., using a known weight of microbial cell catalyst. An IU ofenzyme activity is defined as the amount of enzyme activity required toconvert one micromole of substrate to product per minute.

The temperature of the hydrolysis reaction is chosen to optimize boththe reaction rate and the stability of enzyme catalyst. The temperatureof the reaction may range from just above the freezing point of thereaction mixture (ca. 0° C.) to 65° C., with a preferred range ofreaction temperature of from 5° C. to 45° C. An enzyme catalyst solutionor suspension may be prepared by suspending the unimmobilized orimmobilized cells in distilled water, or in an aqueous reaction mixtureof a buffer that will maintain the initial pH of the reaction between5.0 and 10.0, preferably between 6.0 and 8.0, or by suspending theimmobilized enzyme catalyst in a similar mixture, or by preparing asolution of a cell extract, partially purified or purified enzyme(s), ora soluble form of the immobilized enzymes in a similar mixture. Afterthe nitrile is added and as the reaction proceeds, the pH of thereaction mixture may change due to the formation of an ammonium salt ofthe carboxylic acid from the corresponding nitrile functionality of thealiphatic or aromatic nitrile (when using a combination of nitrilehydratase and amidase enzymes). The reaction can be run to completelyconvert the nitrile with no pH control, or a suitable acid or base canbe added over the course of the reaction to maintain the desired pH.

EXAMPLES

The present invention is further defined in the following Examples thatindicate preferred embodiments of the invention. From the abovediscussion and these Examples, one skilled in the art can ascertain theessential characteristics of this invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the invention to adapt it to various uses and conditions.

In the following Examples, the percent recovery of nitrile, and thepercent yields of the corresponding amide and carboxylic acid productswere based on the initial concentration of nitrile present in thereaction mixture, and were determined by HPLC. Analyses of3-hydroxyvaleronitrile, adiponitrile, butyronitrile, benzonitrile, andmethacrylonitrile were performed by HPLC using a refractive indexdetector in combination with a Supelco LC-18-DB column (15 cm×4.6 mmdiameter) with precolumn at 25° C. and 10 mM acetic acid, 10 mM sodiumacetate in 7.5% methanol in water as eluent at 1.5 mL/min. Analyses forglycolonitrile, acrylonitrile, 3-HPN, 3-HBN, and their correspondingreaction products were performed by HPLC using a Bio-Rad HPX-87H organicacid analysis column (30 cm×7.8 mm dia.) with precolumn at 50° C. and0.010 N H₂SO₄ as eluent at 1 mL/min. Analyses for 3-cyanopyridine,pyrazinecarbonitrile, 2-furancarbonitrile, 2-thiophenecarbonitrile,4-thiazolecarbonitrile and their corresponding reaction products, wereperformed by HPLC using a UV detector at 254 nm in combination with a10-cm×4-mm ID, 5 μm C8 Discovery column (Supelco) with precolumn, 1.0mL/min of 5% CH₃CN/95% 10 mM NaOAc, 10 mM AcOH as solvent, andN,N-dimethylbenzamide as external standard.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Maniatis (supra)and Ausubel (supra).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillip Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds., American Society for Microbiology, Washington,D.C. (1994)) or in Brock, supra.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” meanssecond(s), “min” means minute(s), “h” means hour(s), “d” means day(s),“mL” means milliliters, “L” means liters, “mM” means millimolar, “M”means molar, “mmol” means millimole(s), “rpm” means revolutions perminute, “slpm” means standard liters per minute, “psig” means pounds persquare inch, and “wt” means weight. “HPLC” means high performance liquidchromatography, “ca” means approximately, “O.D.” means optical densityat the designated wavelength, “dcw” means dry cell weight, and “IU”means International Units.

Example 1 Identification of a Genomic DNA Fragment Encoding C.testosteroni 5-MGAM-4D Nitrile Hydratase

Comamonas testosteroni 5-MGAM-4D (ATCC 55744) was grown in LB media at37° C. with shaking. Genomic DNA was prepared using a Puregene DNAIsolation Kit according to the manufacturer (Gentra Systems,Minneapolis, Minn.). A Southern analysis (Southern et al., J. Mol.Biol., 98:503 (1975)) was performed on EcoRI restricted genomic DNAusing Psuedomonas putida NRRL-18668 genes (SEQ ID NOs:1 and 2) encodingnitrile hydratase alpha and beta subunits (U.S. Pat. No. 5,811,286) asprobes. Probe labeling, hybridization, and detection were done using ECLrandom prime labeling and detection systems version 11 according to themanufacturer (Amersham International, Buckinghamshire, UK). The alpha(SEQ ID NO:1) and beta (SEQ ID NO:2) probes each showed positivehybridization to the same 5.7 kb EcoRI DNA fragment.

Example 2 Cloning of a Genomic DNA Fragment Encoding C. testosteroni5-MGAM-4D nitrile hydratase

Genomic DNA from C. testosteroni 5-MGAM-4D was prepared (Example 1),restricted with EcoRI, and subjected to standard agarose gelelectrophoresis. DNA fragments in the size range of approximately 5-7 kbwere isolated and ligated to EcoRI restricted pUC19 (New EnglandBiolabs, Beverly, Mass.). This plasmid library was plated and screenedwith the P. putida NRRL-18668 nitrile hydratase α-subunit gene probe(SEQ ID NO:1). Probe labeling, hybridization and detection were doneusing ECL random prime labeling and detection systems version 11according to the manufacturer (Amersham International). A positivelyhybridizing colony was isolated and determined to contain an insert of5.7 kb (pKP57).

Example 3 Determination of the Nucleotide Sequence of the Genes EncodingC. testosteroni 5-MGAM-4D Nitrile Hydratase

The nucleotide sequence of the pKP57 (EXAMPLE 2) insert was determinedusing an ABI 377-XL DNA sequencer and BigDye Terminator Cycle Sequencingchemistry. A BlastN analysis (Altschul et al., Nucleic Acids Res.,25:3389-3402 (1997)) of the obtained sequence to the GenBank® databaseconfirmed the presence of complete genes encoding nitrile hydratase αand β-subunits and a partial gene encoding amidase. Nucleotide sequencesof the pKP57 insert encoding nitrile hydratase α- and β-subunits aregiven in SEQ ID NO:3 and SEQ ID NO:5, respectively. Deduced amino acidsequences of the pKP57 insert for the α- and β-subunits are given in SEQID NO:4 and SEQ ID NO:6, respectively.

BlastP analysis was conducted using the deduced amino acid sequence forthe nitrile hydratase α- and β-subunits. Results are shown in Table 1.The closest match to the instant amino acid sequence of the nitrilehydratase α-subunit was the nitrile hydratase α-subunit from Pseudomonasputida NRRL-18668 (97.1% identity, 97.1% similarity, E value=10e⁻¹⁰⁸).The closest match to the instant amino acid sequence of the nitrilehydratase β-subunit was the nitrile hydratase β-subunit from Pseudomonasputida NRRL-18668 (82.0% identity, 82.5% similarity, E value=6e⁻⁹²).

Example 4 Production of C. testosteroni 5-MGAM-4D Nitrile Hydratase inE. coli

A DNA fragment encoding the nitrile hydratase alpha and beta subunitsfrom C. testosteroni 5-MGAM-4D (SEQ ID NO:7) was obtained from pKP57 bystandard PCR via a GeneAmp Kit according to the manufacturer (Roche,Branchburg, N.J.) using Primer 1 (SEQ ID NO:8) and Primer 2 (SEQ IDNO:9) and subcloned into pGEM-T (Promega, Madison, Wis.) under controlof the T7 promoter to generate pSW131. E. coli BL21(DE3) (Novagen,Madison, Wis.) was transformed with pSW131 using standard procedures.Growth and induction of E. coli BL21 (DE3) harboring pSW131 was carriedout essentially as recommended by Novagen. Modifications included theaddition of cobalt chloride and sodium citrate at the time of inductionto a final concentration of 0.01 mg/ml and 0.1 mg/ml respectively.Induction was carried out at 30 C for 16 hrs. Production of alpha (23kDa) and beta (24 kDa) proteins was confirmed by standard SDS-PAGEanalysis.

Example 5 Conversion of 3-Hydroxyvaleronitrile (3-HVN) by Transformed E.coli

Growth and induction of E. coli BL21(DE3) cells harboring pSW131 wascarried out as described in EXAMPLE 4. Cells were then harvested bycentrifugation, washed twice in buffer (0.1 M potassium phosphate pH7.0) and suspended at 100 mg wet cells/ml in buffer. The nitrilaseactivity assay mix included cells (50 mg/mL), 3-hydroxyvaleronitrile(0.3 M) and buffer (0.1 M potassium phosphate, pH 7.0) stirred atambient temperature. HPLC analysis demonstrated 17% conversion of 3-HVNto the corresponding amide (3-hydroxyvaleramide) in 15 min.

Example 6 Production of High-Level C. testosteroni 5-MGAM-4D NitrileHydratase Activity in E. coli Requires Downstream Sequence

A plasmid (pSW132; ATCC PTA-5073), containing a DNA fragment encodingthe C. testosteroni 5-MGAM-4D nitrile hydratase alpha and beta subunitsplus 0.9 kb of downstream DNA encoding the accessory protein P7K (SEQ IDNO:11) under control of the T7 promoter was constructed by replacing thesmaller BamHI/PstI fragment in pSW131 with the corresponding BamHI/PstIfragment from pKP57. E. coli BL21(DE3) (Novagen) was transformed withpSW132 using standard procedures. Growth and induction of E. coliBL21(DE3) harboring pSW132 (“E. coli strain SW132”) was carried out asdescribed in EXAMPLE 4, and production of alpha and beta proteins wasconfirmed by standard SDS-PAGE analysis.

A DNA fragment (SEQ ID NO:11) encoding the C. testosteroni 5-MGAM-4Dnitrile hydratase alpha and beta subunits plus 0.9 kb of downstream DNAencoding the accessory protein P7K was also obtained from pKP57 bystandard PCR via a GeneAmp Kit according to the manufacturer (Roche)using Primer 1 (SEQ ID NO:8) and Primer 3 (SEQ ID NO:12) and subclonedinto pTrcHis2-TOPO under control of the trc promoter to generate pSW137in E. coli TOP10 according to the manufacturer (Invitrogen). Growth andinduction of E. coli TOP10 harboring pSW137 was carried out as describedin EXAMPLE 4, and production of alpha and beta proteins was confirmed bystandard SDS-PAGE analysis. Production of the alpha and beta proteins inE. coli BL21(DE3) harboring pSW132 and in E. coli TOP10 harboring pSW137was qualitatively indistinguishable to that obtained from E. coli BL21(DE3) harboring pSW131 (EXAMPLE 4).

Growth and induction of E. coli BL21 (DE3) harboring pSW132 was carriedout as described in EXAMPLE 4. Cells were then harvested bycentrifugation, washed twice in buffer (0.1 M potassium phosphate pH7.0) and suspended at 100 mg wet cells/ml in buffer. The nitrilehydratase activity assay mix included cells (2 mg/mL),3-hydroxyvalerontrile (0.3 M) and buffer (0.1 potassium phosphate, pH7.0) stirred at ambient temperature. HPLC analysis demonstrated 100%conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleramide in 5 min.Similarly, nitrile hydratase activity assay of E. coli TOP10 harboringpSW137 demonstrated 100% conversion of 3-hydroxyvaleronitrile to3-hydroxyvaleramide in 5 min. Comparing these results to those obtainedfrom pSW131 (Example 5) demonstrated the importance of the DNAdownstream of the nitrile hydratase beta gene, in obtaining maximalnitrile hydratase activity (Table 2). The downstream nucleotide sequence(SEQ ID NO:10) contains a small open reading frame, the sequence ofwhich is given in SEQ ID NO:13. The deduced amino acid sequence (calledP7K) is given in SEQ ID NO:14.

TABLE 2 Comparison of Expression Vectors pSW131 and pSW132 % Conversionof Expressed Genetic 0.35 M 3-HVN Expression Components from to 3-HVAmVector C. testosteroni 5-MGAM-4D at Room Temperature PSW131 Nitrilehydratase α- and β-subunits  17% in 15 min PSW132 Nitrile hydratase α-and β-subunits 100% in 5 min plus accessory protein “P7K”

Example 7 Determination of the Nucleotide Sequence of the Gene EncodingC. testosteroni 5-MGAM-4D Amidase

Genomic DNA from C. testosteroni 5-MGAM-4D was prepared (EXAMPLE 1),restricted with PstI, and subjected to Southern analysis using astandard PCR product comprising the first 0.6 kb of the pKP57 (EXAMPLE2) insert as a probe (SEQ ID NO:15). Probe labeling, hybridization anddetection were done using ECL random prime labeling and detectionsystems version 11 according to the manufacturer (AmershamInternational). This probe gave hybridized to a 2.4 kb PstI fragment.Genomic DNA digested with PstI was subjected to standard agarose gelelectrophoresis. DNA fragments in the size range of approximately 2-4 kbwere isolated and ligated into PstI restricted pUC19. This plasmidlibrary was plated and screened with the same 0.6 kb probe (SEQ IDNO:15). Probe labeling, hybridization and detection were done using ECLrandom prime labeling and detection systems version 11 according to themanufacturer (Amersham International). A positively hybridizing colonywas isolated and determined to contain an insert of 2.4 kb (pKP59).

Nucleotide sequencing confirmed that the insert is a DNA fragment thatoverlaps the EcoRI DNA fragment previously cloned (pKP57). Thus, bycombining the nucleotide sequences from pKP57 and pKP59, the completenucleotide sequence for the amidase gene was determined (SEQ ID NO:16).The deduced amidase amino acid sequence is given in SEQ ID NO:17. Thenucleotide sequence of a 7.4 kb DNA fragment from C. testosteroni5-MGAM4D comprising complete coding sequences for amidase and nitrilehydratase is given in SEQ ID NO:18.

BlastP analysis was conducted using the deduced amidase amino acidsequence (SEQ ID NO:17; Table 1). The closest publicly known match wasto that of the amidase from Pseudomonas putida NRRL-18668 (92.3%identity, 92.5% similarity, E value=0).

Example 8 Production of C. testosteroni 5-MGAM-4D Amidase in E. coli

A DNA fragment encoding the amidase from C. testosteroni 5-MGAM-4D wasobtained from genomic DNA by standard PCR via a GeneAmp Kit according tothe manufacturer (Roche), using Primer 4 (SEQ ID NO:19) and Primer 5(SEQ ID NO:20) and subcloned into pGEM-T (Promega) under control of theT7 promoter to generate pKP60. E. coli BL21 (DE3) (Novagen) wastransformed with pKP60 using standard procedures. Growth and inductionof E. coli BL21 (DE3) harboring pKP60 was carried out according toNovagen, and production of amidase protein was confirmed by standardSDS-PAGE analysis

A DNA fragment encoding the amidase from C. testosteroni 5-MGAM-4D wasalso obtained by standard PCR using Primer 6 (SEQ ID NO:21) and Primer 7(SEQ ID NO:22) and subcloned into pTrcHis2 TOPO under control of the trcpromoter to generate pSW133 in E. coli TOP10 according to themanufacturer (Invitrogen). Growth and induction of E. coli TOP10harboring pSW133 was carried out according to Invitrogen, and productionof amidase protein was confirmed by standard SDS-PAGE analysis.

Example 9 Co-Production of C. testosteroni 5-MGAM-4D Nitrile Hydrataseand Amidase in E. coli

A DNA fragment encoding the amidase, NHase alpha and beta, and accessoryprotein P7K from C. testosteroni 5-MGAM-4D (SEQ ID NO:23) was obtainedfrom genomic DNA by standard PCR via a GeneAmp Kit according to themanufacturer (Roche), using Primer 6 (SEQ ID NO:21) and Primer 3 (SEQ IDNO:12), and subcloned into pTrcHis2-TOPO (Invitrogen) under control ofthe Trc promoter to generate pSW136.

Example 10 Fermentation of Escherichia coli SW132 Cells

The production of nitrile hydratase in a 14 L Braun Biostat C fermentor(B. Braun Biotech International Gmbh, Melsungen, Germany) was made inmineral medium with glucose, ammonia, and yeast extract. E. coli strainSW132 (E. coli BL21(DE3) harboring plasmid pSW132 as described inExample 6) was grown in a seed culture for 10 h prior to inoculation ofthe fermentor. IPTG (1 mM) was added to the fermentor at 30-35OD_(λ=550) and cells were harvested 5 h after IPTG addition.Fermentation protocol: vessel medium was prepared in an initial batch of7.5 L containing 32 g KH₂PO₄, 8.0 g MgSO₄*7H₂O, 8.0 g (NH₄)₂SO₄, 50 gyeast extract, and 10 mL Mazu DF204 antifoam (BASF Corporation, MountOlive, N.J.). Following sterilization, 369 g glucose solution (60% w/w),160 mL trace element solution (Table 3), and 100 mg/L ampicillin wereadded. NH₄OH (40% w/v) and 20% w/v H₂SO₄ were used for pH control. Theset points for agitation, aeration, pH, pressure, dissolved oxygenconcentration (DO), and temperature are described in Table 4 below. Thedissolved oxygen concentration was controlled at 25% of air saturationwith the agitation to rise first with increase oxygen demand and theaeration to follow. The 500 mL seed culture was grown in a 2 L flask at36° C., 300 rpm for 10 h to an OD_(λ=550) of >2.0. In the fermentor atculture densities of 20-30OD additional AMP was added to 100 mg/L. IPTGwas added to 1 mM at culture densities of 30-35OD. Glucose feed wasstarted at <5 g/L and the scheduled rates are described in Table 5.Glucose feed rate was reduced if glucose accumulated above 2 g/L. Fivehours after IPTG addition the cells were chilled to 5-10° C. andharvested by centrifugation; 490 g (wet cells) was harvested. Thekinetics of growth and nitrile hydratase production are presented inTable 6.

TABLE 3 Trace elements solution: Chemical Concentration g/L Citric acid10.0 CaCl₂*2H₂O 1.50 FeSO₄*7H₂O 5.00 ZnSO₄*7H₂O 0.39 CuSO₄*5H₂O 0.38CoCl₂*6H₂O 0.20 MnCl₂*4H₂O 0.30

TABLE 4 Fermentation Run Conditions Initial Set-Point Minimum MaximumStirrer (rpm) 400 400 850 Airflow (slpm) 2 2 16 pH 6.8 6.8 6.8 Pressure(psig) 0.5 0.5 0.5 DO 25% 25% 25% Temp. C. 36 36 36

TABLE 5 Glucose feed protocol Time, h Rate (g/min) 0-2 0.39 2-8 0.788-End 0.60

TABLE 6 The kinetics of growth and nitrile hydratase production NitrileHydratase Glucose Production Time, h OD_(λ=550) (g/L) (U/g dry cell wt.)2.9 5.8 26.2 4.9 15.7 20.2 6.3 28 8.5 9157 8.1 44 6 10220 11.2 57.6 0.0410888

Example 11 Hydration of Nitriles to Corresponding Amides byUnimmobilized E. coli SW132 Cells

To a 20-mL reaction vessel equipped with magnetic stirring was added0.04, 0.4, 2.0, or 5.0 mmol of acrylonitrile, methacrylonitrile,3-hydroxypropionitrile, 3-hydroxybutyronitrile, 3-hydroxyvaleronitrile,butyronitrile, adiponitrile, benzonitrile, or glycolonitrile anddistilled, deionized water was added to adjust the final volume of themixture to 3.0 mL. To the reaction vessel was next added 1.0 mL of anaqueous suspension of 0.44-8.8 mg dry cell weight (dcw)/mL of E. coliSW132 cells (prepared as described in Example 10) in 0.10 M potassiumphosphate buffer (pH 7.0, except for glycolonitrile, which was run at pH6.0), and the mixture was stirred at 25° C. Samples (0.100 mL) of thereaction mixture were mixed with 0.400 mL of water, and then 0.200 mL ofthe diluted sample was either (a) mixed with 0.200 mL of 0.200 M sodiumbutyrate (acrylonitrile and 3-hydroxyvaleronitrile HPLC standard), 0.200M N-ethylacetamide (methacrylonitrile HPLC standard), 0.200 M isobutyricacid (butyronitrile and 3-hydroxypropionitrile HPLC standard), or 0.200M malonic acid (3-hydroxybutyronitrile HPLC standard) in water, or (b)measured against a calibration curve for product at 100% nitrileconversion (5-cyanovaleramide, adipamide, benzamide, glycolamide). Theresulting mixture was centrifuged, and the supernatant analyzed by HPLC.

All reactions produced only the amide as the hydration product at 100%conversion of nitrile, with no hydrolysis of the nitrile to thecorresponding carboxylic acid.

TABLE 7 Hydration of Nitriles to Corresp. Amides by E. coli SW132 Cellsconc. mg time Amide yield Nitrile (M) dcw/mL (h) amide (%) Acrylonitrile0.50 2.2 3 acrylamide 100 Acrylonitrile 1.25 8.8 5 acrylamide 98Methacrylonitrile 0.50 2.2 20 methacrylamide 100 3-hydroxypropionitrile0.50 2.2 3 3-hydroxypropionamide 100 3-hydroxybutyronitrile 0.50 4.4 43-hydroxybutryamide 99 3-hydroxyvaleronitrile 0.51 2.2 13-hydroxyvaleramide 99 Butyronitrile 0.50 2.2 16 butyramide 100Adiponitrile 0.50 0.44 1.5 5-cyanovaleramide 938 adipamide Adiponitrile0.50 4.4 1 adipamide 100 Benzonitrile 0.010 2.2 2 benzamide 99Glycolonitrile 0.10 2.2 2 glycolamide 63

Example 12 Hydration of 3-Hydroxyvaleronitrile using aPartially-Purified Protein Extract of E. coli SW132 Cells

E. coli SW132 cells (0.4874 g) were suspended in 2.0 mL of cold breakingbuffer consisting of 1 mM DTT and 0.1 mM PMSF in 0.1 M potassiumphosphate buffer (pH 7). The suspension (200 mg wet cell weight/mL) wasloaded in a French Pressure Mini Cell, and the cells were ruptured at16000-17000 psi. Cell debris was removed from the resulting mixture bycentrifugation at 38000 RCF for 15 min. Approximately 1.68 mL of extractsupernatant was recovered, having a nitrile hydratase activityequivalent to a 199 mg wet cell weight/mL cell suspension.

To a 20-mL reaction vessel equipped with magnetic stirring was added 0.4mL of the E. coli SW132 extract supernatant, 0.6 mL of deionized water,and 3 mL of 0.667 M 3-hydroxyvaleronitrile in water. The mixture wasstirred at 25° C. Samples (0.100 mL) of the reaction mixture were mixedwith 0.100 mL of water, and then 0.200 mL of the diluted sample wasmixed with 0.200 mL of 0.200 M sodium butyrate (external standard) andanalyzed by HPLC. After 120 min, the yield of 3-hydroxyvaleramide was99% at 100% conversion of 3-hydroxyvaleronitrile.

Example 13 Comparison of Thermal Stability of Nitrile Hydratase fromComamonas testosteroni 5-MGAM-4D and Pseudomonas putida 5B Cells

A 44 mg dry cell weight/mL suspension of either E. coli SW30 wet cells(having active nitrile hydratase from Pseudomonas putida 5B) or E. coliSW132 wet cells (having active nitrile hydratase from Comamonastestosteroni 5-MGAM-4D) in 0.50 M phosphate buffer was heated to 50° C.in a water bath. At predetermined times, aliquots of the 50° C. cellsuspensions were rapidly cooled to 25° C. in a water bath, and thesesuspensions were assayed for remaining nitrile hydratase activity byadding 1.0 mL aliquots of the heated/cooled cell suspensions withstirring to 3.0 mL of 0.667 M 3-hydroxyvaleronitrile in water at 25° C.Samples (0.100 mL) of the reaction mixture were withdrawn atpredetermined times and mixed with 0.100 mL of water, then 0.200 mL ofthe diluted sample was mixed with 0.200 mL of 0.200 M sodium butyrate(external standard), centrifuged, and the supernatant and analyzed byHPLC. The rate of hydration of 3-hydroxyvaleronitrile in each reactionwas determined, and remaining nitrile hydratase specific activity of thecells calculated. The nitrile hydratase specific activity and % ofenzyme recovered for E. coli SW30 and E. coli SW132, respectively, as afunction of time at 50° C. is listed in Table 8, below.

TABLE 8 Comparison of E. coli SW30 and E. coli SW132 Nitrile HydrataseThermostability. SW132 Time SW30 Nitrile Nitrile Nitrile Nitrile At 50°C. Hydratase Hydratase Hydratase Hydratase (min) (IU/g dcw) Recovery (%)(IU/g dcw) Recovery (%) 0 103 100 7752 100 30 1.6 1.6 6940 90 60 0 06437 83

Example 14 Comparison of Comamonas testosteroni 5-MGAM-4D andPseudomonas putida 5B Nitrile Hydratase for Hydration of Acrylonitrileto Acrylamide

To a 20-mL reaction vessel (equipped with magnetic stirring) was added0.270 g (5.1 mmol) of acrylonitrile and a suspension of either 107.6 mgdry cell weight E. coli SW30 wet cells (expressing the active nitrilehydratase from Pseudomonas putida 5B) or 4.41 mg dry cell weight E. coliSW132 wet cells (expressing the active nitrile hydratase from Comamonastestosteroni 5-MGAM-4D) in a total volume of 9.664 mL of 0.10 Mpotassium phosphate buffer (pH 7.0); the amount of dry cell weightpresent in each reaction was chosen to provide ca. equivalent nitrilehydratase activities (IU/mL) in the reaction mixtures. The finalconcentration of acrylonitrile was 0.51 M. The mixture was stirred at25° C. Samples (0.200 mL) of the reaction mixture were mixed with 0.200mL of 0.200 M sodium butyrate (HPLC standard) in water, and 0.020 mL of6 N acetic acid. The resulting sample was centrifuged, and thesupernatant analyzed by HPLC. The reaction time, % acrylonitrileconversion, and % yield of acrylamide are listed in Table 9. Forreactions using E. coli SW30 as biocatalyst, a loss of nitrile hydrataseactivity was observed over the course of the reaction, and incompleteconversion of nitrile was obtained at extended reaction times.

TABLE 9 Comparison of E. coli SW30 and E. coli SW132 in AcrylonitrileHydration Reactions E. coli mg time acrylonitrile conv. acrylamide yieldConstruct dcw/mL (h) (%) (%) SW30 10.8 1.5 42 38 SW30 10.8 20 80 82 SW3010.8 45 96 97 SW132 0.44 1.5 100 98

Example 15 Comparison of Comamonas testosteroni 5-MGAM-4D andPseudomonas putida 5B Nitrile Hydratase for Hydration of3-Hydroxyvaleronitrile to 3-Hydroxyvaleramide

To a 20-mL reaction vessel (equipped with magnetic stirring) was added3.0 mL of a solution of 0.204 g (2.0 mmol) of 3-hydroxyvaleronitrile indistilled, deionized water and a 1.0 mL suspension of 44 mg dry cellweight of either E. coli SW30 wet cells (expressing the active nitrilehydratase from Pseudomonas putida 5B) or E. coli SW132 wet cells(expressing the active nitrile hydratase from Comamonas testosteroni5-MGAM-4D) in 0.10 M potassium phosphate buffer (pH 7.0). The finalconcentration of 3-hydroxyvaleronitrile was 0.50 M. The mixture wasstirred at 25° C. Samples (0.100 mL) of the reaction mixture were mixedwith 0.100 mL of water, 0.200 mL of 0.200 M sodium butyrate (HPLCstandard) in water, an 0.020 mL of 6 N HCl. The resulting mixture wascentrifuged, and the supernatant analyzed by HPLC. The reaction time, %3-hydroxyvaleronitrile conversion, and % yield of 3-hydroxyvaleramideare listed in Table 10. For reactions using SW30 as biocatalyst, a lossof nitrile hydratase activity was observed over the course of thereaction, and incomplete conversion of nitrile was obtained at extendedreaction times.

TABLE 10 Comparison of E. coli SW30 and E. coli SW132 in 3-Hydroxyvaleronitrile Reactions 3-hydroxy- 3-hydroxy- E. coli mg Timevaleronitrile valeramide Construct dcw/mL (h) conv. (%) yield (%) SW3011 1 12 12 SW30 11 7 42 40 SW30 11 24 61 61 SW132 11 0.25 100 100

Example 16 Hydration of 3-Hydroxyvaleronitrile to 3-Hydroxyvaleramide byE. coli SW137

To a 4-mL reaction vessel (equipped with magnetic stirring) was added0.75 mL of an aqueous solution containing 0.404 M 3-hydroxyvaleronitrileand 0.25 mL of a suspension of 18 mg dry cell weight E. coli SW137 wetcells (prepared as described in Example 6) in 0.10 M potassium phosphatebuffer (pH 7.0). The E. coli SW137 cells express the polypeptide havingnitrile hydratase activity from Comamonas testosteroni 5-MGAM-4D. Thefinal concentration of 3-hydroxyvaleronitrile was 0.303 M. The mixturewas stirred at 25° C. Samples (0.100 mL) of the reaction mixture weremixed with 0.100 mL of water, 0.200 mL of 0.200 M sodium butyrate (HPLCstandard) in water, an 0.020 mL of 6 N HCl. The resulting mixture wascentrifuged, and the supernatant analyzed by HPLC. After 10 min, theconversion of 3-hydroxyvaleronitrile was 100%, and the yield of3-hydroxyvaleramide was 100%.

Example 17 Immobilization of Escherichia coli SW132 Cells in CalciumCross-Linked Alginate

Into a 250-mL media bottle (equipped with magnetic stir bar andcontaining 59.7 g of distilled, deionized water at 50° C.) was slowlyadded 3.30 g of FMC BioPolymer Protanal® LF 10/60 alginate with rapidstirring. The mixture was heated to 75-80° C. with rapid stirring untilthe alginate was completely dissolved, and the resulting solution cooledto 25° C. in a water bath. To the alginate suspension was added 40.8 gof Escherichia coli SW132 wet cell paste (22% dry cell weight) and 16.2mL of distilled water with stirring. The cell/alginate mixture was addeddropwise by syringe to 640 mL of 0.20 M calcium acetate buffer (pH 7.0)at 25° C. with stirring. After stirring for 2 h, the buffer was decantedfrom the resulting beads (82 g), which were resuspended in 200 mL of0.20 M calcium acetate buffer (pH 7.0) at 25° C. With stirring, 4.10 gof 25 wt % glutaraldehyde (GA) in water was added and the beads mixedfor 1.0 h at 25° C. To the suspension was then added 16.4 g of 12.5 wt %polyethylenimine (PEI) (BASF Lupasol® PR971L, average molecular weightca. 750,000) in water, and the beads mixed for an additional 1 h at 25°C. The GA/PEI-crosslinked beads were then washed twice with 250 mL of0.05 M calcium acetate buffer (pH 7.0) at 25° C., and stored in thissame buffer at 5° C.

Example 18 Hydration of Nitriles (0.50 M to 3.0 M) to CorrespondingAmides by Alginate-Immobilized Escherichia coli SW132 Cells inConsecutive Batch Reactions with Biocatalyst Recycle

Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer(temperature-controlled at 25° C. or 35° C.) with a recirculatingtemperature bath) was placed 4.0 g of GA/PEI-crosslinked Escherichiacoli SW132 cell/alginate beads prepared as described in Example 17. Tothe reaction vessel was added 0.2 mL of 0.20 M calcium acetate buffer(pH 7.0, 2.0 mM final calcium ion concentration in reaction mixture),10, 20, 40 or 60 mmol of acrylonitrile, methacrylonitrile, or3-hydroxy-valeronitrile, and the final volume of the reaction mixtureadjusted to 20 mL by the addition of distilled, deionized water. Themixture was stirred at 25° C. or 35° C. Samples (0.100 mL) of thereaction mixture were mixed with 0.400 mL of water, and then 0.200 mL ofthe diluted sample was mixed with 0.200 mL of 0.200 M sodium butyrate(acrylonitrile and 3-hydroxyvaleronitrile HPLC external standard) or0.200 M N-ethylacetamide (methacrylonitrile HPLC external standard) inwater. The resulting mixture was centrifuged, and the supernatantanalyzed by HPLC.

At the completion of the reaction (100% conversion of nitrile), theproduct mixture was decanted from the biocatalyst beads, and additionaldistilled, deionized water, 0.2 mL of 0.20 M calcium acetate buffer (pH7.0, 2.0 mM final calcium ion concentration in reaction mixture) and 10,20, 40 or 60 mmol of acrylonitrile, methacrylonitrile, or3-hydroxy-valeronitrile mixed with the reaction heel (immobilized-cellcatalyst and remaining product mixture from the first reaction) at 25°C. or 35° C. At the completion of the second reaction, the productmixture was decanted and a third reaction performed as before. Thereaction time, product yield for acrylamide, methacrylamide, or3-hydroxyvaleramide, and the percent recovered biocatalyst activity foreach recycle reaction is listed in Table 11 below.

TABLE 11 Hydration of Nitriles (0.50 M to 3.0 M) to Corresponding Amidesby Immobilized E. coli SW132 Cells in Consecutive Batch Reactions withBiocatalyst Recycle Recovered Bio- Amide catalyst Conc. Temp. Rxn TimeYield Activity Nitrile (M) (° C.) # (h) (%) (%) Acrylonitrile 0.53 25 10.18 100 100 2 0.18 100 94 3 0.12 100 112 Acrylonitrile 1.02 25 1 0.25100 100 2 0.25 100 118 3 0.25 100 124 acrylonitrile 2.05 25 1 0.5 100100 2 0.5 100 95 3 0.5 100 100 acrylonitrile 3.04 25 1 0.75 100 100 20.83 100 98 3 0.5 100 106 acrylonitrile 1.06 35 1 0.15 100 100 2 0.25100 107 3 0.15 100 110 methacrylonitrile 1.00 25 1 1.0 100 100 2 1.0 100106 3 1.5 99 103 3- 1.05 25 1 0.75 100 100 hydroxyvaleronitrile 2 0.75100 93 3 0.75 100 84 3- 2.05 25 1 1.5 100 100 hydroxyvaleronitrile 2 2.0100 102 3 2.0 100 93

Example 19 Immobilization of E. coli SW132 Cells in Carrageenan

Into a 250 mL media bottle equipped with magnetic stir bar andcontaining 54.6 g of water at 50° C. is slowly added 2.88 g ofkappa-carrageenan (FMC RG300) with rapid stirring. The mixture is heatedto 75-80° C. with rapid stirring until the carrageenan is completelydissolved, and the resulting solution cooled to 55-56° C. (ca. 52° C.gelling temperature) in a thermostated water bath. A suspension of 18.6g of E. coli SW132 wet cell paste (22.0% dry cell wt) in 19.7 g of 0.35M sodium phosphate buffer (pH 7.3) is heated to 50° C. for 15 min, thenadded to the carrageenan solution at 55-56° C. with stirring. Thecell/carrageenan mixture is immediately added slowly to 383 mL ofsoybean oil at 50° C. with stirring using an overhead stirrer. Aftercell/carrageenan droplets of the desired size are produced in the oil bycontrolling the stirring rate, the temperature of the oil is reduced to40-42° C. to gel the droplets, and the oil decanted from the resultingbeads. The beads are washed with 150 mL of 0.1 M potassium bicarbonatebuffer (pH 7.0), then suspended in 182 mL of this same buffer, and 1.9 gof 25 wt % glutaraldehyde in water is added and the beads mixed for 1 hat 25° C. To the mixture is then added 7.6 g of 12.5 wt %polyethylenimine (BASF Lupasol PR971 L) average Mw ca. 750,000) inwater, and the beads mixed for 1 h at 25° C. The beads are then washedtwice with 0.30 M ammonium bicarbonate (pH 7.0), and stored in this sameat 5° C.

Example 20 Hydration of Acrylonitrile by Carrageenan-Immobilized E. coliSW132 Cells

Into a 50-mL jacketed reaction vessel equipped with an overhead stirrer(temperature-controlled at 35° C. with a recirculating temperature bath)is placed 4.0 g of GA/PEI-crosslinked E. coli SW132 cell/carrageenanbeads prepared as described in Example 19. To the reaction vessel isthen added 1.06 g of acrylonitrile (1.0 M final concentration), thefinal volume of the reaction mixture adjusted to 20 mL by the additionof distilled, deionized water, and the mixture stirred at 35° C. Samples(0.100 mL) of the reaction mixture are mixed with 0.400 mL of water, andthen 0.200 mL of the diluted sample is mixed with 0.200 mL of 0.200 Msodium butyrate (HPLC external standard) in water. The resulting mixtureis centrifuged, and the supernatant analyzed by HPLC. At completeconversion of acrylonitrile, there is a quantitative yield ofacrylamide.

Example 21 Hydration of Acrylonitrile Using Immobilized NitrileHydratase from Partially-Purified Protein Extract of E. coli SW132 Cells

Into a 25-mL Erlenmeyer flask is weighed 1.0 g of oxirane acrylic beads(Sigma). To the flask is then added ca. 7.5 mL of a solution containingpotassium phosphate buffer (50 mM, pH 8.0), and the oxirane acrylicbeads suspended in the buffer by briefly mixing the contents of theflask. After cessation of mixing, the beads settle to the bottom of theflask, and the fine particles which float to the top of the mixture areremoved by pipette, along with as much of the supernatant which can beremoved without disturbing the settled beads. This washing procedure isrepeated a second time. To the flask is then added 1.0 mL of the E. coliSW132 cell extract supernatant described in Example 12 and the finalvolume of the mixture adjusted to 10 mL with additional potassiumphosphate buffer. The resulting mixture is mixed on a rotary platformshaker for 16 h at 25° C. The mixture is then transferred to achromatography column equipped with a fritted bed support, and theimmobilized nitrile hydratase washed three times with 10 mL of potassiumphosphate buffer and stored at 5° C. in this same buffer.

Into a 50-mL jacketed reaction vessel equipped with an overhead stirrer(temperature-controlled at 35° C. with a recirculating temperature bath)is placed 1.0 g of immobilized E. coli SW132 nitrile hydratase preparedas described above. To the reaction vessel is then added 1.06 g ofacrylonitrile (1.0 M final concentration), the final volume of thereaction mixture adjusted to 20 mL by the addition of distilled,deionized water, and the mixture stirred at 35° C. Samples (0.100 mL) ofthe reaction mixture are mixed with 0.400 mL of water, and then 0.200 mLof the diluted sample is mixed with 0.200 mL of 0.200 M sodium butyrate(HPLC external standard) in water. The resulting mixture is centrifuged,and the supernatant analyzed by HPLC. At complete conversion ofacrylonitrile, there is a quantitative yield of acrylamide.

Example 22 Hydration of 3-Cyanopyridine (0.5 M) to Nicotinamide byUnimmobilized E. coli SW132 Cells

To a 15-mL polypropylene centrifuge tube was added 3.73 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2660 gof 3-cyanopyridine. The final concentration of 3-cyanopyridine was 0.501M. The reaction mixture was mixed on a rotating platform at 23° C. After15 minutes, 7.50 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard) was added to thereaction, the resulting mixture centrifuged, and a 0.100 mL of thesupernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC.The conversion of 3-cyanopyridine was 100%, and the yields ofnicotinamide and nicotinic acid were 100% and 0%, respectively.

Example 23 Hydration of 3-Cyanopyridine (1.0 M) to Nicotinamide byUnimmobilized E. coli SW132 Cells

To a 15-mL polypropylene centrifuge tube was added 3.47 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 55.2 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.5339 gof 3-cyanopyridine. The final concentration of 3-cyanopyridine was 1.00M. The reaction mixture was mixed on a rotating platform at 23° C. After30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard) was added to thereaction, the resulting mixture centrifuged, and a 0.100 mL of thesupernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC.The conversion of 3-cyanopyridine was 100%, and the yields ofnicotinamide and nicotinic acid were 99% and 0%, respectively.

Example 24 Hydration of 3-Cyanopyridine Using a Partially-PurifiedProtein Extract of E. coli SW132 Cells

E. coli SW132 cells (0.5377 g, (prepared as described in Example 10))were suspended in 5.377 mL of cold breaking buffer consisting of 1 mMDTT and 0.1 mM PMSF in 0.50 mM potassium phosphate buffer (pH 7.0). Thesuspension (100 mg wet cell weight/mL) was loaded in a French PressureMini Cell, and the cells were ruptured at 16000-17000 psi (approximately110.3-117.2 megapascal (Mpa)). Cell debris was removed from theresulting mixture by centrifugation at 38000 RCF for 15 min.Approximately 4.20 mL of extract supernatant was recovered, having anitrile hydratase activity equivalent to a 100 mg wet cell weight/mL(22.06 mg dry cell weight/mL) cell suspension.

To a 15-mL polypropylene centrifuge tube was added 3.73 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of an E. coli SW132 cellextract suspension prepared as described above, and 0.2626 g of3-cyanopyridine. The final concentration of 3-cyanopyridine was 0.494 M.The reaction mixture was mixed on a rotating platform at 23° C. After 15min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard) was added to thereaction, the resulting mixture centrifuged, and a 0.100 mL of thesupernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC.The conversion of 3-cyanopyridine was 100%, and the yields ofnicotinamide and nicotinic acid were 100% and 0%, respectively.

Example 25 Hydration of Pyrazinecarbonitrile (0.5 M) to Pyrazinamide byUnimmobilized E. coliSW132 Cells

To a 15-mL polypropylene centrifuge tube was added 3.73 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2694 gof pyrazinecarbonitrile. The final concentration of pyrazinecarbonitrilewas 0.512 M. The reaction mixture was mixed on a rotating platform at23° C. After 15 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30M N,N-dimethylbenzamide (HPLC external standard) was added to thereaction, the resulting mixture centrifuged, and a 0.100 mL of thesupernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC.The conversion of pyrazinecarbonitrile was 100%, and the yields ofpyrazinamide and pyrazinecarboxylic acid were 100% and 0%, respectively.

Example 26 Hydration of Pyrazinecarbonitrile (1.0 M) to Pyrazinamide byUnimmobilized E. coli SW132 Cells

To a 15-mL polypropylene centrifuge tube was added 3.47 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 55.2 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.5330 gof pyrazinecarbonitrile. The final concentration of pyrazinecarbonitrilewas 1.00 M. The reaction mixture was mixed on a rotating platform at 23°C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard) was added to thereaction, the resulting mixture centrifuged, and a 0.100 mL of thesupernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC.The conversion of pyrazinecarbonitrile was 100%, and the yields ofpyrazinamide and pyrazinecarboxylic acid were 100% and 0%, respectively.

Example 27 Hydration of 2-Furancarbonitrile (0.5 M) to2-Furancarboxamide by Unimmobilized E. coli SW132 Cells

To a 15-mL polypropylene centrifuge tube was added 3.78 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2381 gof 2-furancarbonitrile. The final concentration of 2-furancarbonitrilewas 0.506 M. The reaction mixture was mixed on a rotating platform at23° C. After 30 min, 7.50 mL of 95:5 acetonitrile/water containing 0.30M N,N-dimethylbenzamide (HPLC external standard) was added to thereaction, the resulting mixture centrifuged, and a 0.100 mL of thesupernatant mixed with 0.900 mL of acetonitrile and analyzed by HPLC.The conversion of 2-furancarbonitrile was 100%, and the yields of2-furancarboxamide and 2-furancarboxylic acid were 99% and 0%,respectively.

Example 28 Hydration of 2-Thiophenecarbonitrile (0.3 M) to2-Thiophenecarboxamide by Unimmobilized E. coli SW132 Cells

To a 15-mL polypropylene centrifuge tube was added 3.86 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.1691 gof 2-thiophenecarbonitrile. The final concentration of2-thiophencarbonitrile was 0.307 M. The reaction mixture was mixed on arotating platform at 27° C. After 30 min, 7.50 mL of 95:5acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLCexternal standard) was added to the reaction, the resulting mixturecentrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL ofacetonitrile and analyzed by HPLC. The conversion of2-thiophenecarbonitrile was 99.5%, and the yields of2-thiophenecarboxamide and 2-thiophenecarboxylic acid were 98% and 0%,respectively.

Example 29 Hydration of 4-Thiazolecarbonitrile (0.5 M) to4-Thiazolecarboxamide by Unimmobilized E. coli SW132 Cells

To a 15-mL polypropylene centrifuge tube is added 3.70 mL of 50 mMpotassium phosphate buffer (pH 7.0), 1.0 mL of a suspension of 22.1 mgdry cell weight E. coli SW132 wet cells (prepared as described inExample 10) in 50 mM potassium phosphate buffer (pH 7.0), and 0.2754 gof 4-thiazolecarbonitrile. The final concentration of4-thiazolecarbonitrile is 0.500 M. The reaction mixture is mixed on arotating platform at 25° C. After 30 min, 7.50 mL of 95:5acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLCexternal standard) is added to the reaction, the resulting mixturecentrifuged, and a 0.100 mL of the supernatant mixed with 0.900 mL ofacetonitrile and analyzed by HPLC. The conversion of4-thiazolecarbonitrile is 100%, and the yields of 4-thiazolecarboxamideand 4-thiazolecarboxylic acid are 100% and 0%, respectively.

Example 29 Hydration of 3-Cyanopyridine (1.0 M) to Nicotinamide byAlginate-Immobilized Escherichia coli SW132 Cells in Consecutive BatchReactions with Biocatalyst Recycle at 25° C.

Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer(temperature-controlled at 25° C. with a recirculating temperature bath)was placed 1.0 g of GA/PEI-crosslinked Escherichia coli SW132cell/alginate beads prepared as described in Example 17. To the reactionvessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mMfinal calcium ion concentration in reaction mixture), 16.67 mL ofdistilled, deionized water, and 2.124 g of 3-cyanopyridine. The finalconcentration of 3-cyanopyridine was 1.00 M in 20 mL of reactionmixture. Samples (0.100 mL) of the reaction mixture were mixed with0.400 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of theresulting solution mixed with 0.900 mL of acetonitrile and analyzed byHPLC. After 24 h, the conversion of 3-cyanopyridine was 100%, and theyields of nicotinamide and nicotinic acid were 100% and 0%,respectively. The initial reaction rate for production of nicotinamide,measured during the first 30 min of reaction, was 7.65 mM/minute.

At the completion of the reaction (100% conversion of nitrile), theproduct mixture was decanted from the biocatalyst beads, and 0.2 mL of0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ionconcentration in reaction mixture), 16.56 mL of distilled, deionizedwater, and 2.127 g of 3-cyanopyridine was added to the catalyst beads inthe reaction vessel. The final concentration of 3-cyanopyridine was 1.06M in 20 mL of reaction mixture. Samples (0.100 mL) of the reactionmixture were mixed with 0.400 mL of 95:5 acetonitrile/water containing0.30 M N,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL ofthe resulting solution mixed with 0.900 mL of acetonitrile and analyzedby HPLC. After 23 h, the conversion of 3-cyanopyridine was 100%, and theyields of nicotinamide and nicotinic acid were 100% and 0%,respectively. The initial reaction rate for production of nicotinamide,measured during the first 30 min of reaction, was 4.60 mM/minute.

Example 30 Hydration of 3-Cyanopyridine (1.0 M) to Nicotinamide byAlginate-Immobilized Escherichia coli SW132 Cells in Consecutive BatchReactions with Biocatalyst Recycle at 10° C.

Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer(temperature-controlled at 10° C. with a recirculating temperature bath)was placed 4.0 g of GA/PEI-crosslinked Escherichia coli SW132cell/alginate beads prepared as described in Example 17. To the reactionvessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mMfinal calcium ion concentration in reaction mixture), 13.63 mL ofdistilled, deionized water, and 2.127 g of 3-cyanopyridine. The finalconcentration of 3-cyanopyridine was 1.00 M in 20 mL of reactionmixture. Samples (0.100 mL) of the reaction mixture were mixed with0.400 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of theresulting solution mixed with 0.900 mL of acetonitrile and analyzed byHPLC. After 1 h, the conversion of 3-cyanopyridine was 100%, and theyields of nicotinamide and nicotinic acid were 100% and 0%,respectively. The initial reaction rate for production of nicotinamide,measured during the first 30 min of reaction, was 23.4 mM/minute.

At the completion of the reaction (100% conversion of nitrile), theproduct mixture was decanted from the biocatalyst beads, and 0.2 mL of0.20 M calcium acetate buffer (pH 7.0, 2.0 mM final calcium ionconcentration in reaction mixture), 13.14 mL of distilled, deionizedwater, and 2.125 g of 3-cyanopyridine was added to the catalyst beads inthe jacketed reaction vessel at 10° C. The final concentration of3-cyanopyridine was 1.00 M in 20 mL of reaction mixture. Samples (0.100mL) of the reaction mixture were mixed with 0.400 mL of 95:5acetonitrile/water containing 0.30 M N,N-dimethylbenzamide (HPLCexternal standard), and a 0.100 mL of the resulting solution mixed with0.900 mL of acetonitrile and analyzed by HPLC. After 1 h, the conversionof 3-cyanopyridine was 94%, and the yields of nicotinamide and nicotinicacid were 94% and 0%, respectively. The initial reaction rate forproduction of nicotinamide, measured during the first 30 min ofreaction, was 18.5 mM/minute.

Example 31 Hydration of 3-Cyanopyridine (3.0 M) to Nicotinamide byAlginate-Immobilized Escherichia coli SW132 Cells in Consecutive BatchReactions with Biocatalyst Recycle at 25° C.

Into a 50-mL jacketed reaction vessel (equipped with an overhead stirrer(temperature-controlled at 25° C. with a recirculating temperature bath)was placed 2.0 g of GA/PEI-crosslinked Escherichia coli SW132cell/alginate beads prepared as described in Example 17. To the reactionvessel was added 0.2 mL of 0.20 M calcium acetate buffer (pH 7.0, 2.0 mMfinal calcium ion concentration in reaction mixture), 11.42 mL ofdistilled, deionized water, and 6.3648 g of 3-cyanopyridine. The finalconcentration of 3-cyanopyridine was 3.00 M in 20 mL of reactionmixture. Samples (0.100 mL) of the reaction mixture were mixed with1.400 mL of 95:5 acetonitrile/water containing 0.30 MN,N-dimethylbenzamide (HPLC external standard), and a 0.100 mL of theresulting solution mixed with 0.900 mL of acetonitrile and analyzed byHPLC. After 48 h, the conversion of 3-cyanopyridine was 100%, and theyields of nicotinamide and nicotinic acid were 100% and 0%,respectively.

1. A method for hydrating methacrylonitrile to methacrylamide comprisinga) contacting methacrylonitrile, under suitable reaction conditions,with a catalyst having nitrile hydratase activity from Comamonastestosteroni 5-MGAM-4D.
 2. The method of claim 1, further comprising b)recovering the methacrylamide produced in step a).
 3. A method ofhydrating acrylonitrile to acrylamide comprising a) contactingacrylonitrile, under suitable reaction conditions, with a catalysthaving nitrile hydratase activity from Comamonas testosteroni 5-MGAM-4D.4. The method of claim 3, further comprising b) recovering theacrylamide formed in step a).
 5. The method of claims 1 or 3, whereinthe catalyst is in the form of whole cells, permeabilized microbialcells, one or more components of a microbial cell extract, partiallypurified enzyme, or purified enzyme.
 6. The method of claim 5, whereinthe catalyst is immobilized on or in a soluble or insoluble support. 7.The method of claim 6, wherein the catalyst is immobilized in alginateor carrageenan.
 8. A method for hydrating 3-cyanopyridine tonicotinamide comprising a) contacting 3-cyanopyridine, under suitablereaction conditions, with a catalyst having nitrile hydratase activityfrom Comamonas testosteroni 5-MGAM-4D.
 9. The method of claim 8, furthercomprising b) recovering the nicotinamide produced in step a).
 10. Amethod of hydrating pyrazinecarbonitrile to pyrazinecarboxamidecomprising a) contacting pyrazinecarbonitrile, under suitable reactionconditions, with a catalyst having nitrile hydratase activity fromComamonas testosteroni 5-MGAM-4D.
 11. The method of claim 10, furthercomprising b) recovering the pyrazinecarboxamide formed in step a). 12.The method of claims 8 or 10, wherein the catalyst is in the form ofwhole cells, permeabilized microbial cells, one or more components of amicrobial cell extract, partially purified enzyme, or purified enzyme.13. The method of claims 8 or 10, wherein the catalyst is immobilized onor in a soluble or insoluble support.
 14. The method of claims 8 or 10,wherein the catalyst is immobilized in alginate or carrageenan.